Many kinds of cancer cells possess defects in the pathways of apoptosis. Members of the Bcl-2 family of proteins play pivotal roles in regulating apoptosis and possess at least 1 of 4 conserved Bcl-2-Homology (BH) domains, designated, BH1, BH2, BH3, and BH4. The BH3 domain is the only one (1) present in pro-apoptotic "BH3-only" molecules and plays important roles in protein-protein interactions and apoptosis by regulating dimerization and multimerization of the Bcl-2 family members. To date, approximately 21 human genes have been found that encode proteins belonging to the Bcl-2 family, 14 of which are BH3-only pro-apoptotic proteins. This shows that the BH3-only proteins are important and possess overlapping, complementary and/or specific roles in different types of cells in apoptosis. Therefore, the overall goals of this study are to identify and characterize novel BH3-only proteins from the human proteome and to delineate functions of the newly identified proteins in cancer cell death. We hypothesized that there are additional genes encoding BH3-only proteins in the human genome that interact with other members of the Bcl-2 family and/or other molecules in regulating apoptosis in cell type and/or signaling-specific manners. We further hypothesize that some of the newly identified BH3-only proteins possess additional (i.e., non-apoptogenic) functions in normal physiology. In the preliminary studies, we utilized the bioinformatic data-mining approach to identify 3 novel BH3-only proteins. The candidate genes, apolipoprotein L6 (ApoL6), ApoL.1 and BH3- Only Member B (BOMB), were cloned and functionally expressed in cancer models. By transient expression analysis, we showed that ApoL6, ApoL1 and BOMB promoted cell death in p53-null HCT116 cells. Furthermore, by constructing and characterizing an inducible ApoL6 gene in another p53-null cancer model, DLD-1, we found that expression of ApoL6 induced a mitochondria-mediated apoptosis in DLD-1 cells. These hypotheses will be tested further with 5 specific aims. Deciphering functions of the novel BH3-only proteins and their interacting molecules should enhance our understanding, and potential utilization, of BH3-only proteins and lipid-mediated apoptosis in treatment of cancer.